Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition - Oxford Academic Journals
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9310–9326 Nucleic Acids Research, 2021, Vol. 49, No. 16 Published online 13 August 2021
https://doi.org/10.1093/nar/gkab693
Structural and mechanistic insights into the Artemis
endonuclease and strategies for its inhibition
Yuliana Yosaatmadja1,† , Hannah T. Baddock2,† , Joseph A. Newman1 , Marcin Bielinski3 ,
Angeline E. Gavard1 , Shubhashish M.M. Mukhopadhyay1 , Adam A. Dannerfjord1 ,
Christopher J. Schofield3 , Peter J. McHugh 2,* and Opher Gileadi 1,*
1
Centre for Medicines Discovery, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK, 2 Department
of Oncology, MRC-Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK and 3 The
Downloaded from https://academic.oup.com/nar/article/49/16/9310/6350767 by guest on 12 December 2021
Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, Chemistry Research Laboratory,
University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
Received December 22, 2020; Revised July 20, 2021; Editorial Decision July 25, 2021; Accepted August 11, 2021
ABSTRACT INTRODUCTION
Artemis (SNM1C/DCLRE1C) is an endonuclease that Nucleases catalyse the hydrolysis of the phosphodiester
plays a key role in development of B- and T- bonds in nucleic acids and are broadly classified as
lymphocytes and in dsDNA break repair by non- exonucleases or endonucleases. Exonucleases are often
homologous end-joining (NHEJ). Artemis is phos- non sequence-specific, while endonucleases can be fur-
phorylated by DNA-PKcs and acts to open DNA hair- ther grouped into sequence-specific endonucleases, such as
restriction enzymes, and structure-selective endonucleases
pin intermediates generated during V(D)J and class- (1). Artemis (SNM1C or DCLRE1C), along with SNM1A
switch recombination. Artemis deficiency leads to (DCLRE1A) and SNM1B (Apollo or DCLRE1B), are hu-
congenital radiosensitive severe acquired immune man nucleases that are members of the extended structural
deficiency (RS-SCID). Artemis belongs to a super- family of metallo--lactamase (MBL) fold enzymes (2,3).
family of nucleases containing metallo--lactamase The N-terminal region of Artemis has a core MBL fold
(MBL) and -CASP (CPSF-Artemis-SNM1-Pso2) do- (aa 1–170, 319–361) with an inserted -CASP (CPSF73,
mains. We present crystal structures of the catalytic Artemis, SNM1 and PSO2) domain (aa 170–318) (4). -
domain of wildtype and variant forms of Artemis, in- CASP domains are present within the larger family of eu-
cluding one causing RS-SCID Omenn syndrome. The karyotic nucleic acid processing MBLs and confer both
catalytic domain of the Artemis has similar endonu- DNA/RNA binding and nuclease activity (2,5). The C-
clease activity to the phosphorylated full-length pro- terminal region of Artemis (aa 362–692) mediates protein-
protein interactions, contains post translational modifica-
tein. Our structures help explain the predominantly tion (PTM) sites, directs subcellular localization, and may
endonucleolytic activity of Artemis, which contrasts modulate catalysis (6–9).
with the predominantly exonuclease activity of the Although SNM1A, SNM1B and Artemis have similar
closely related SNM1A and SNM1B MBL fold nucle- structures of their core catalytic domains, each has distinct
ases. The structures reveal a second metal binding functions and selectivities. While SNM1A and SNM1B are
site in its -CASP domain unique to Artemis, which exclusively 5 to 3 exonucleases, Artemis is an endonucle-
is amenable to inhibition by compounds including ase (7,9), although a minor 5 to 3 exonuclease activity
ebselen. By combining our structural data with that has been reported (10). Human SNM1A localizes to sites
from a recently reported Artemis structure, we were of DNA damage, can digest past DNA damage lesions in
able model the interaction of Artemis with DNA sub- vitro, and is involved in inter-strand crosslink (ICL) repair
strates. The structures, including one of Artemis with (11–13). SNM1B is a shelterin-associated protein required
for resection at newly-replicated leading-strand telomeres
the cephalosporin ceftriaxone, will help enable the to generate the 3 -overhang necessary for telomere loop (t-
rational development of selective SNM1 nuclease in- loop) formation and telomere protection (14–16). SNM1A
hibitors. and SNM1B prefer ssDNA substrates in vitro, with an abso-
lute requirement for a free 5 -phosphate (3,17). By contrast,
* To
whom correspondence should be addressed. Tel: +44 7880553524; Email: ogileadi@gmail.com
Correspondence may also be addressed to Peter J. McHugh. Email: peter.mchugh@imm.ox.ac.uk
†
The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
C The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research, 2021, Vol. 49, No. 16 9311
Artemis preferentially cleaves hairpins and DNA junctions, cloning (LIC) (38)) into the baculovirus expression vector
although it is able to process ssDNA substrates (18–20). pBF-6HZB (GenBank™ accession number KP233213.1),
The key roles of Artemis and related DSB repair en- which combines an N-terminal His6 sequence,the Z-basic
zymes in both programmed V(D)J recombination and non- tag and a TEV protease cleavage site for efficient purifica-
programmed c-NHEJ DSB repair, make them attractive tion. Site-directed mutagenesis was carried out using an in-
targets for treatment of cancer, either on their own or in verse PCR method whereby an entire plasmid is amplified
combination with chemo- or radiotherapy. The Artemis using complementary mutagenic primers (oligonucleotides)
endonuclease activity is responsible for hairpin opening with minimal cloning steps (39), using the Herculase II Fu-
in variable (diversity) joining (V(D)J) recombination (21) sion DNA Polymerase (Agilent) for amplification and the
and contributes to end-processing in the canonical non- KLD enzyme mix (NEB).
homologous end joining (c-NHEJ) DNA repair path-
way (22–25). V(D)J recombination is initiated by recog-
nition and binding of recombination-activating gene pro- Expression and purification of WT and mutant Artemis with
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teins (RAG1 and RAG2) to recombination signal sequences IMAC (aa 1-362)
(RSSs) adjacent to the V, D and J gene segments. Upon Baculovirus generation was performed as described (3). Re-
binding, the RAG proteins induce double-strand breaks combinant proteins were produced by infecting Sf9 cells at
(DSBs) and create a hairpin at the coding ends (26–28). The 2 × 106 cells/ ml with 1.5 ml of P2 virus for WT and 3 ml
Ku heterodimer recognizes the DNA double-strand break of P2 virus for mutants respectively. Infected Sf9 cells were
and recruits DNA-dependent protein kinase catalytic sub- harvested 70 h after infection by centrifugation (900 × g,
unit (DNA-PKcs) and Artemis to mediate hairpin open- 20 min). The cell pellets were resuspended in 30 ml/l ly-
ing (19). Following hairpin opening, the NHEJ machin- sis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10
ery containing the XRCC4/XLF(PAXX)/DNA-Ligase IV mM imidazole, 5% (v/v) glycerol and 1 mM TCEP), snap
complex is recruited to catalyse the processing and ligation frozen in liquid nitrogen, then stored at −80◦ C for later
reactions at the DNA ends (22,29,30). V(D)J recombination use.
is an essential process in antibody maturation (18,31,32). Thawed cell aliquots were lysed by sonication. The lysates
Mutations in the Artemis gene cause aberrant hairpin were centrifuged (40 000 × g, 30 min); the supernatant was
opening, resulting in severe combined immune deficiency passed through a 0.80 m filter (Millipore), then loaded
(RS-SCID), with sensitivity to ionizing radiation due to im- onto an immobilized metal affinity chromatography col-
pairment of the predominant DSB repair pathway in mam- umn (IMAC) (Ni-NTA Superflow Cartridge, Qiagen) equi-
malian cells, NHEJ (19,21,29), and another form of SCID librated in lysis buffer. The column was washed with lysis
(Omenn syndrome) associated with hypomorphic Artemis buffer, then eluted using a linear gradient to elution buffer
mutations (33,34). Artemis loss-of-function mutations of- (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM imidazole,
ten comprise large deletions in the first four exons or non- 5% (v/v) glycerol and 1 mM TCEP). Protein-containing
sense founder mutation, as found in Navajo and Apache fractions were pooled and passed through an ion exchange
Native Americans (35). In addition, missense mutations and column (HiTrap® SP FF GE Healthcare Life Sciences) pre-
in-frame deletions in the highly conserved residues such as equilibrated in the SP buffer A (25 mM HEPES pH 7.5, 300
H35, D165 and H228 can also abolish Artemis’ protein mM NaCl, 5% (v/v) glycerol and 1 mM TCEP). Protein was
function (36). eluted using a linear gradient to SP buffer B (SP buffer A
We present high-resolution crystal structures of the cat- with 1 M NaCl), and fractions containing the ZB-tagged
alytic core of Artemis (aa 1–361) containing both MBL and Artemis were identified by electrophoresis.
a -CASP domains. The structures reveal that Artemis pos- Artemis containing fractions were pooled and dialysed
sesses a second metal binding site in its -CASP domain, overnight at 4◦ C in SP buffer A, supplemented with re-
not present in SNM1A and SNM1B, that resembles classi- combinant tobacco etch virus (TEV) protease to cleavage
cal Cys2 His2 zinc finger motifs. Based on our data and an- the His6 -ZB tag. The protein was loaded into an ion ex-
other recently reported Artemis structure (37), we present a change column (HiTrap® SP FF GE Healthcare Life Sci-
model for Artemis DNA binding. The model is compared ences), pre-equilibrated in the SP buffer A. The protein
with models of DNA binding from related nucleases, re- was eluted using a linear gradient to SP buffer B; frac-
vealing distinct features that define a role for Artemis in tions containing tag-free Artemis were identified by elec-
the end-joining reaction. Following development of an as- trophoresis. Artemis-containing fractions from the SP col-
say suitable for screening, we identified -lactam containing umn elution were combined and concentrated to 1 ml using
Artemis inhibitors, one of which, the cephalosporin ceftri- a 30 kDa MWCO centrifugal concentrator. The protein was
axone, was characterized crystallographically, providing in- then loaded on to a Superdex 75 increase 10/300 GL equi-
formation useful for structure-based design of inhibitors. librated with SEC buffer (25 mM HEPES pH 7.5, 300 mM
NaCl, 5% (v/v) glycerol, 2 mM TCEP).
MATERIALS AND METHODS Mass spectrometric analysis of the purified proteins re-
vealed masses of 41716.5, 41650.5, 41672.2, 41639.9 Da
Cloning and site directed mutagenesis of WT and mutant
for WT, H35A, D37A and H35D proteins, respectively.
Artemis (aa 1-362)
The calculated masses are 41715.09, 41649.2, 41671.2 and
Constructs encoding the Artemis MBL--CASP domain 14639.2, respectively, all within 1.5 Da of the measured
(WT and mutant) were cloned (using ligation independent masses.
9312 Nucleic Acids Research, 2021, Vol. 49, No. 16
Expression and purification of WT truncated Artemis cat- 60 psig, drying gas at 350◦ C and drying gas flow rate at 12
alytic domain without IMAC (aa 1–362) l/min. The instrument ion optic voltages were as follows:
fragmentor 250 V, skimmer 60 V and octopole RF 250 V.
The truncated Artemis protein was produced in a similar
manner except that the first purification step (IMAC) was
omitted. The tight binding of the ZB-tagged protein to the Protein crystallization and soaking
ion exchange column and the early elution of the tag-free
protein allowed to achieve effective purification. After the Artemis (PDB: 6TT5) was crystallized using the sitting drop
second ion exchange step, the protein was further puri- vapour diffusion method by mixing 50 nl protein with 50 nl
fied by size exclusion chromatography (Highload® 16/200 crystallization solution comprising 0.2 M ammonium chlo-
Superdex® 200). ride, 20% (v/v) PEG 3350. Crystals appeared after 2 weeks
and reached maximum size within 3 weeks. The crystals
were soaked in cryoprotectant solution (mother liquor sup-
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Cloning, expression, and purification of full-length WT plemented with 20% (v/v) ethylene glycol), then flash frozen
Artemis (aa 1-692) in liquid nitrogen.
The non-IMAC purified Artemis (PDB: 7AF1) was crys-
The full-length Artemis encoding construct was cloned into
tallized in a similar manner, with addition of 20 nl of crystal
pFB-CT10HF-LIC, a baculovirus transfer vector contain-
seed solution obtained from previous crystallization exper-
ing a C-terminal His10 and FLAG tag, using ligation inde-
iment. Crystals were grown in a solution comprising 0.25
pendent cloning (LIC) (38). pFB-CT10HF-LIC was a gift
M ammonium chloride and 30% (v/v) PEG 3350 at 4◦ C.
from Nicola Burgess-Brown (Addgene plasmid # 39191;
Crystals appeared after one day and reached a maximum
http://n2t.net/addgene:39191; RRID: Addgene 39191).
size within one week.
The baculovirus mediated expression of the full length
Artemis variants (mutants H33A and H35D) were crys-
DCLRE1C/ Artemis gene was performed in a manner sim-
tallized using the sitting drop vapour diffusion method by
ilar to that for the truncated protein using 3.0 ml of P2 virus
mixing 50 nl protein with 50 nl crystallization solution com-
to infect Sf9 cells at 2 × 106 cells/ml.
prising 0.1 M sodium citrate pH 5.5, 20% PEG 3350; the
Cell harvesting and the initial IMAC purification steps
D37A variant was crystalized in 0.2 M ammonium acetate,
were performed as described for the catalytic domain. Fol-
0.1 M bis-Tris pH 5.5, 25% PEG 3350. All Artemis vari-
lowing IMAC purification and TEV cleavage overnight in
ants were crystalized in the presence of 20 nl of crystal seed
dialysis buffer (50 mM HEPES pH 7.5, 0.5 M NaCl, 5%
solution obtained from previous crystallization experiment.
glycerol and 1 mM TCEP) the protein was passed through
Crystals grew after one day at 4◦ C. and reached maximum
a 5 ml Ni-sepharose column to trap the tag and other metal-
size within 1 week.
binding contaminants; the flowthrough fractions were col-
lected. The Artemis protein was then concentrated using
a centrifugal concentrator (Centricon, MWCO 30 kDa) Data collection and refinements
before loading on a Superdex S200 HR 16/60 gel filtra-
tion column in dialysis buffer. Fractions containing purified Data were collected at Diamond Light Source beamlines
Artemis protein were pooled and concentrated to 10 mg/ml. I04, I03 and I24. Diffraction data were processed using
DIALS (40) and structures were solved by molecular re-
placement using PHASER (41) and the PDB coordinates
Electrospray ionization mass spectrometry 5Q2A. Model building and the addition of water molecules
Reversed-phase chromatography was performed prior to were performed in COOT (42) and structures refined us-
mass spectrometry using an Agilent 1290 uHPLC system ing REFMAC (43). Data collection and refinement statis-
(Agilent Technologies Inc., Palo Alto, CA, USA). Con- tics are given in Table I. The X-ray fluorescence data was
centrated protein samples were diluted to 0.02 mg/ml in collected at Diamond Light Source I03 (6TT5) using 100%
0.1% aqueous formic acid and 50 l was injected on to transmission and 12.7 eV, and I24 (7AF1) using 1% trans-
a 2.1 mm × 12.5 mm Zorbax 5um 300SB-C3 guard col- mission and 12.8 eV (Supplementary Figure S1).
umn housed in a column oven set at 40◦ C. The solvent sys-
tem used consisted of 0.1% aqueous formic acid in ultra-
Generation of 3 -radiolabelled substrates
high purity water (Millipore) (solvent A) and 0.1% aque-
ous formic acid in methanol (LC–MS grade, Chromasolve) 10 pmol of single-stranded DNA (Eurofins MWG Operon,
(solvent B). Initial chromatography conditions were 90% A Germany) was labelled with 3.3 pmol of ␣-32 P-dATP
and 10% B and a flow rate of 1.0 ml/min. A linear gradi- (Perkin Elmer) by incubation with terminal deoxynu-
ent from 10% B to 80% B was applied over 35 s. Elution cleotidyl transferase (TdT, 20 U; ThermoFisher Scientific),
then proceeded isocratically at 95% B for 40 s followed by at 37◦ C for 1 h. This solution was then passed through a
equilibration at initial conditions for a further 15 s. Protein P6 Micro Bio-Spin chromatography column (BioRad), and
intact mass was determined using a 6530-electrospray ion- the radiolabeled DNA was annealed with the appropriate
ization quadrupole time-of-flight mass spectrometer (Agi- unlabeled oligonucleotides (1:1.5 molar ratio of labelled to
lent Technologies Inc., Palo Alto, CA, USA). The instru- unlabeled oligonucleotide) (Supplementary Table S1 for se-
ment was configured with the standard ESI source and op- quences) by heating to 95◦ C for 5 min and gradual cooling
erated in positive ion mode. The ion source was operated to below 30◦ C in annealing buffer (10 mM Tris–HCl; pH
with the capillary voltage at 4000 V, nebulizer pressure at 7.5, 100 mM NaCl, 0.1 mM EDTA).
Nucleic Acids Research, 2021, Vol. 49, No. 16 9313
Gel-based nuclease assays nickel ion at the M1 site and a partial occupancy zinc ion at
the M2 site (PDB: 6TT5) (Figure 1I); the identification of a
Standard nuclease assays were carried out in reactions con-
nickel ion (at M1 and/or M2 sites) was supported by XRF
taining 20 mM HEPES–KOH, pH 7.5, 50 mM KCl, 10
(Supplementary Figure S1). A similar overall metal ion
mM MgCl2 , 0.05% (v/v) Triton X-100, 5% (v/v) glycerol
coordination pattern has been observed with other mem-
(final volume: 10 l), and the indicated concentrations of
bers of the family, such as SNM1A and SNM1B/Apollo
Artemis. Reactions were started by addition of substrate
(3,11).
(10 nM), incubated at 37◦ C for the indicated time, then
The overall fold of the catalytic core of Artemis is similar
quenched by addition of 10 l stop solution (95% for-
to that of SNM1A and SNM1B (2 Å backbone RMSD);
mamide, 10 mM EDTA, 0.25% (v/v) xylene cyanole, 0.25%
it has all the key structural characteristics of human MBL
(v/v) bromophenol blue) with incubation at 95◦ C for 3 min.
fold nucleases, with the di-metal containing active site at the
Reaction products were analysed by 20% denaturing
interface between the MBL and -CASP domains (Figure
polyacrylamide gel electrophoresis (made from 40% solu-
1A). Its MBL domain (Figure 1A, B) has the typical ␣/-
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tion of 19:1 acrylamide:bis-acrylamide, BioRad) and 7 M
/␣ sandwich MBL fold (45) and contains the conserved
urea) in 1× TBE (Tris–borate–EDTA) buffer. Electrophore-
motifs 1–4 (Figure 1C and D, Supplementary Figure S2)
sis was carried out at 700 V for 75 min; gels were subse-
typical of the MBL superfamily and motifs A–C typical of
quently fixed for 40 min in a 50% methanol, 10% acetic acid
the -CASP fold family (2,4,29,46).
solution, and dried at 80◦ C for 2 h under a vacuum. Dried
Motifs 1–4 (Figure 1D–J) are responsible for metal ion
gels were exposed to a Kodak phosphor imager screen and
coordination in both DNA and RNA processing MBL fold
scanned using a Typhoon 9500 instrument (GE).
enzymes (4). As observed in structures of SNM1A and
SNM1B, Artemis can coordinate one or two metal ions in
Fluorescence-based nuclease assay its active site. In the di-zinc complexed structure, one zinc
ion (M1) is coordinated by four residues (His33, His35,
The protocol of Lee et al. (44) was adapted for structure-
His115 and Asp116) and two water molecules (H2 O 506
specific endonuclease activity. A ssDNA substrate was uti-
and 611) in an octahedral manner (Figure 1H). The second
lized containing a 5 FITC-conjugated T and a 3 BHQ-1
zinc ion (M2) was refined with 30% occupancy and is coor-
(black hole quencher)-conjugated T (Supplementary Table
dinated by Asp37, His38 and Asp136 and two waters. The
S1). As the FITC and BHQ-1 are located proximal to one
low occupancy of the second zinc ion, together with the two
another, prior to endonucleolytic incision, the intact sub-
conformations (0.5 occupancy for each conformation) ob-
strate does not fluoresce. Following endonucleolytic inci-
served for Asp37 (Figure 1I and J) suggest that the M2 site
sion by SNM1C/Artemis, uncoupling of FITC from BHQ-
binds a metal ion less tightly than the M1 site, consistent
1 causes an increase in fluorescence. Inhibitors (at increas-
with studies on other human MBL fold nucleases (3, Bad-
ing concentrations) were incubated with Artemis (50 nM)
dock et al. 2021)
for 10 min at room temperature, before starting the reaction
The structure of human SNM1A has been solved with a
by adding the DNA substrate (25 nM). Assays were carried
single zinc ion coordinated in its active site (Figure 1E) (3).
out in a 384-well format, in 25 l reaction volumes. The
By contrast, SNM1B structures solved with a bound AMP
buffer was the same as for the gel-based nuclease assays.,
[Baddock et al., 2021 accompanying paper] show two metal
Fluorescence spectra were measured using a PHERAstar
ions, both positioned to coordinate the phosphate group of
FSX (excitation: 495 nm; emission: 525 nm) with readings
the AMP (Figure 1F). In summary, the coordination for the
taken every 150 s, for 35 min, at 37◦ C.
M1 site zinc ion involves three histidines, one aspartate, and
either water molecules or a phosphate oxygen of the sub-
RESULTS strate. The M2 site metal ion is more weakly coordinated
in the SNM1 family, with one histidine and two aspartates,
Human Artemis (SNM1C or DCLRE1C) has a core cat-
with the remaining three positions occupied by water or a
alytic fold similar to SNM1A and Apollo/SNM1B
phosphate oxygen of the substrate. Thus, the presence of the
The catalytic domain of Artemis (aa 3–361) fused to the substrate at the active site may help promote full metal ion
basic His6 -Zb tag, which confers tight binding to cation occupancy at the M2 site. We propose that Artemis coordi-
exchange columns, was produced in baculovirus-infected nates a phosphate group of its substrate in a similar manner
Sf9 cells. The protein was purified using immobilized metal to that proposed for SNM1B.
affinity chromatography (IMAC) on a nickel-Sepharose col- A structure of human CPSF-73 (PDB: 2I7V), an MBL
umn as the initial step. Subsequent preparations were per- RNA processing nuclease, has been solved with two active
formed without the use of IMAC, to avoid introduction of site bound zinc ions, an adjacent sulphate, and a bridging
Ni2+ ions. Artemis crystals were grown and diffracted to water molecule proposed to play an important role in hy-
1.6 Å resolution (Table 1); the structure was solved using a drolysis (Figure 1G) (47). The CPSF-73 structure shows
structure of SNM1A (PDB: 5Q2A) as a molecular replace- that the two zinc ions are coordinated in a very similar ge-
ment model. The resultant structure (PDB: 7AF1; space ometry with the human MBL DNA processing enzymes
group P1) contains a single molecule in the asymmetric unit, (48,49). A striking difference between the MBL RNA and
with two active site zinc ions (i.e. at both M1 and M2 sites) DNA nucleases is that the second metal ion (M2) in the
(Figure 1). Copurifying metal ions were identified using X- RNA processing nucleases is coordinated by an additional
ray fluorescence (XRF) analysis. With the protein purified histidine (His418 for CPSF-73) (47), which has no counter-
using IMAC, the structure was refined with a full occupancy part in the DNA processing enzymes.
9314 Nucleic Acids Research, 2021, Vol. 49, No. 16
Table 1. Data collections and refinement statistics
6TT5 7APV 7ABS (DNA
PDB ID (Ni and Zn) 7AF1 (2 Zn) 7AFS (D37A) 7AFU (H33A) 7AGI (H35D) (Ceftriaxone) bound)
Data Collection and processing
Diffraction Source DLS (I04) DLS (I24) DLS (I03) DLS (I03) DLS (I03) DLS (I03) APS 17-IDD
Wavelength (Å) 0.979 0.976 0.976 0.976 0.976 0.976 1.00
Space group P1 P1 P1 P1 P1 P1 P21212
Cell dimensions
a, b, c (Å) 35.87, 47.99, 35.75, 35.91, 48.06, 35.97, 47.90, 35.97, 48.05, 35.88, 48.10, 72.81, 111.00,
48.25 47.97, 48.15 48.21 48.37 48.44 48.25 55.17
◦
α, β, γ ( ) 82.61, 76.37, 82.68, 76.35, 82.89, 76.43, 82.51, 75.94, 82.43,76.01, 82.76, 76.29, 90.00, 90.00,
85.98 85.81 86.38 87.73 87.33 86.30 90.00
Resolution (Å) * 47.55–1.50 47.53–1.70 35.35–1.70 35.53–1.56 47.62–1.70 47.69–1.95 34.5–1.97
(1.53–1.50) (1.73–1.70) (1.73–1.70) (1.59–1.56) (1.73–1.70) (2.00–1.95) (2.02–1.97)
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Rmerge (%)* 6.0 (64.4) 13.3 (79.5) 5.3 (53.2) 4.9 (31.5) 4.8 (23.2) 11.6 (65.0) 5.0 (82.8)
I/σ (I) 13.4 (3.2) 5.8 (2.0) 11.7 (2.2) 11.3 (2.6) 13.4 (3.8) 8.6 (2.8) 16 (2.4)
Completeness (%) 94.7 (66.4) 97.4 (95.6) 97.3 (96.0) 94.2 (63.7) 97.4 (95.4) 98.0 (97.2) 99.6 (99.6)
Multiplicity 3.6 (3.2) 3.5 (3.5) 3.6 (3.7) 3.6 (3.3) 3.7 (3.8) 3.5 (3.6) 6.4 (6.6)
Refinements
Resolution (Å) 47.55–1.50 47.53–1.70 47.66–1.70 35.53–1.56 47.62–1.70 47.96–1.95 40–1.97
No. of reflections 44 746 31 282 34 470 39 478 31 745 21 086 30 565
Rwork 0.17 0.19 0.18 0.19 0.19 0.18 0.22
Rfree 0.19 0.21 0.22 0.21 0.21 0.23 0.28
No. of atoms
Protein 2927 2989 2957 2985 3122 2975 2923
Water 224 144 163 255 164 107 105
Zinc/nickle 3 3 2 1 1 2 3
Ethylene glycol 24 28 12 44 32 16 –
DNA – – – – – – 253
Ceftriaxone – – – – 1 –
B-factors 16.3 19.3 25.9 17.5 18.4 22.6 56
r.m.s. deviations
Bond length (Å) 0.003 0.008 0.008 0.006 0.007 0.01 0.007
Bond angles (◦ ) 1.23 1.33 1.29 1.28 1.32 1.5 1.47
Data in parentheses is for the high-resolution shell.
The structure of Artemis reveals a novel zinc-finger like motif only His254 seen in all three enzymes. However, the four
in the -CASP domain residues that form the zinc-finger like motif are conserved
in Artemis across the animal kingdom (from humans to
Proteins with a -CASP fold form a distinct sub-group
sponges), implying functional importance (Supplementary
in the MBL- superfamily that specifically act on nucleic
Figure S3). Consistently, substitution of His228 and His254
acids (2). Artemis’ -CASP domain (aa 156–384) is inserted
(H228N and H254L), two of the zinc coordinating residues
within the MBL fold sequence between ␣-helices 6 and 7
in the -CASP domain of Artemis, cause RS-SCID in hu-
(Figure 1). Notably, a second metal ion binding site, unique
mans (4,36,56). Patients with these inherited mutations suf-
to Artemis, with similarity to the canonical Cys2 His2 zinc-
fer from impaired V(D)J recombination, leading to under-
finger motif, is present in its -CASP domain (50,51). Many
developed B and T lymphocytes. The full-length H254A
DNA binding proteins, including transcription factors and
Artemis variant is unable to carry out V(D)J recombina-
DNA repair factors (including those involved in NHEJ),
tion in vivo and has no discernible endonucleolytic activity
possess a Cys2 His2 zinc finger motif that stabilizes the DNA
in vitro (4).
binding domain (50–53). A typical Cys2 His2 zinc coordi-
nating finger (Figure 2A) has a ␣ motif, wherein the zinc
ion is coordinated between an ␣-helix and two antiparal- Comparison of Artemis structures leading to a model for
lel -sheets. Hydrophobic residues located at the sides of DNA binding
the zinc coordination site enable specific binding of the zinc
finger in the major groove of the DNA (50,51,54,55). Simi- During our work two Artemis structures (PDB: 6WO0
lar to the canonical Cys2 His2 zinc finger motif, the zinc ion and 6WNL) similar to our structure (PDB: 7AF1) (back-
coordination in Artemis’ -CASP domain adopts a tetrahe- bone RMSDs of 0.48 and 0.54 Å, respectively) were re-
dral geometry, with coordination by two cysteine (Cys256, ported (37), with identical relative positioning of the MBL
Cys272) and two histidine (His228, His254) residues (Fig- and -CASP domains (Figure 3A and Supplementary
ure 2B). However, in the case of Artemis the metal ion co- Figure S4A). The only significant difference was that whilst
ordination site is sandwiched between two -sheets instead we refined our structure PDB: 7AF1 with two zinc ions in
of an ␣-helix and two antiparallel -sheets. the active site, both of the crystal forms reported by Karim
All but one of the residues in the zinc-finger-like mo- et al. (37) were modelled with a single active site zinc ion
tif (His228, Cys256 and Cys227) are distinct to Artemis (Zn1), reinforcing the proposal of weaker metal ion bind-
relative to SNM1A/B (Supplementary Figure S2), with ing at the Zn2/M2 site.
Nucleic Acids Research, 2021, Vol. 49, No. 16 9315
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Figure 1. Overall fold of the SNM family and the active site views of human MBL/-CASP nuclease fold enzymes. (A) Cartoon representation of the
structure of human SNM1C/ Artemis; the active site containing MBL domain in pink; the -CASP domain (white) contains a novel zinc-finger like motif.
The three zinc ions are represented by grey spheres; the N- and C-termini are marked in red lettering. (B) Topology of Artemis; -strands are represented
as arrows and ␣-helices as cylinders. The MBL domain (pink) has the typical ␣/-/␣ sandwich fold, with the -CASP domain (white) inserted between
the small helix ␣8 and ␣9. (C) Overlay of structures of the human SNM1A, SNM1B and SNM1C. (D) Cartoon sequence alignment for SNM1A, SNM1B
and Artemis, showing the MBL and -CASP domains. Each of the 4 highly conserved motifs (1–4), are in red. Motif 1 = Asp, motif 2 = 3 His and 1 Asp
(HxHxDH), motif 3 = His and motif 4 = Asp. (E) SNM1A as purified has a single octahedrally coordinated zinc (grey) (PDB: 5AHR). (F) Active site
view of SNM1B/ Apollo (PDB: 7A1F) with a nickel ion (green, M1) and an iron ion (orange, M2) with a coordinating AMP molecule. (G) Active site
view of the human RNA processing enzyme CPSF73 (PDB: 2I7V) containing a sulphate ion and a water bridging (asterisk*) the two zincs. One zinc (M2)
is coordinated by an additional histidine residue (His 418) which has no counterpart in SNM1 proteins. (H) Artemis (PDB: 7AF1) purified in the absence
of IMAC has two zincs (grey) in its active site. A water/hydroxide shared (asterisk*) between the two metals is the proposed nucleophile for the hydrolytic
reaction. (I) Active site of human Artemis (PDB: 6TT5) purified with IMAC. A nickel is present in the first metal coordination site (M1) and a zinc in the
second (M2). (J) The overlay of Artemis active site views (H) (PDB: 7AF1) and (I) (PDB: 6TT5).
9316 Nucleic Acids Research, 2021, Vol. 49, No. 16
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Figure 2. Comparison of a novel zinc-finger like motif in the -CASP domain of SNM1C/Artemis with a canonical zinc-finger motif. (A) Cartoon repre-
sentation of a classical Cys2 His2 zinc-finger motif (green) from the transcription factor SP1F2 (PDB code: 1SP2). This has a ␣ fold, where two Cys- and
two His-residues are involved in zinc ion coordination and the sidechains of three conserved hydrophobic residues are shown. (B) The -CASP region of
Artemis has a novel zinc-finger like motif. The inset shows the four residues (two His and two Cys) coordinating the zinc ion (grey). The Fo – Fc electron-
density map (scaled to 2.5 in PyMOL) surrounding the zinc ion before it was included in refinement is shown.
A striking aspect of the reported (37) structures is that ing close contacts with the metal ion centre in a manner
both crystal forms were obtained in the presence of DNA consistent with the proposed catalytic mechanism (Figure
and were reported to require DNA for their growth; the 3). The sequence of the longest strand corresponds to the
crystals showed a fluorescence signal supporting the pres- 10-nucleotide cy-5 labelled strand (cy5-GCGATCAGCT)
ence of DNA (the oligonucleotides used contained a cya- with residual density at the 5 -end that may be attributed
nine dye fluorophore), yet neither of the models presented to the cyanine fluorophore which we did not include in our
contain DNA. The authors referred to some broken stack- model. The complementary strand used in crystallization
ing electron density in 6WNL in a solvent channel and a was 13-nucleotides long and was intended to produce a 5 -
patch of unsolved density approaching the active site in overhang, but only two bases and three phosphates could be
6WO0, but state that the DNA ‘did not bind to the protein located in the density. The abrupt manner in which the elec-
in a physiological way, and likely bound promiscuously to tron density apparently disappears from either end of this
promote crystallization’ (37). We re-examined these struc- strand suggests that this is the product of a cleavage reac-
tures looking closely at the residual electron density. For tion, although it is possible that remaining nucleotides are
the 6WNL structure we found evidence for a distorted du- not located due to disorder.
plex DNA of around 13 base pairs which we propose may The analysis of electron density at this site is complicated
be the product of duplex annealing of the oligonucleotide by the proximity to a crystallographic 2-fold symmetry axis,
used for crystallization (a semi-palindromic 13-mer that was which brings a symmetry copy of the DNA molecule into a
designed to form a hairpin with phospho-thioate linkages position where atoms partially overlap and the extended 5
in the single-stranded region) (Supplementary Figure S4B). strands form a pseudo duplex (Supplementary Figure S5A).
For this structure, we are in general agreement with Karim The occupancy of the entire DNA molecule is thus lim-
et al. that the DNA does not appear to make meaningful ited to 0.5, and the lower occupancy is reflected in the elec-
interactions with the protein that inform on the mechanism tron density map which requires a lower contour level than
of nuclease activity, although this mode of association with would usually be applied (Supplementary Figure S5B). Af-
DNA may be relevant to alternative binding modes relating ter carefully building and refining the afore-described DNA
to higher order complexes containing Artemis. bound model, significant positive electron density was re-
For the PDB: 6WO0 structure we were able to confidently vealed for the second metal ion (M2 site) which we in-
build a DNA molecule that contacts the Artemis active site cluded in the model with the same occupancy (0.5) as the
in a manner that we believe to be relevant to the Artemis DNA. Our model was refined to similar crystallographic
nuclease activity. The model contains an 8-nucleotide 5 - R-factors as 6WO0 and has been deposited with PDB ac-
single-stranded extension with a short 2-base pair region of cession number 7ABS (refinement statistics are given in
duplex DNA that reaches into the Artemis active site, mak- Table I).Nucleic Acids Research, 2021, Vol. 49, No. 16 9317
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Figure 3. Electron density of a DNA molecule in previously reported structures of Artemis. (A) Analysis of the reported (PDB: 6WO0) Artemis structure
re-refined with DNA present (PDB code: 7ABS). The Fo – Fc electron density map (contoured at 0.6 in pymol) for the DNA is in green mesh. (B)
The 7ABS structure (aquamarine) overlayed with the apo-Artemis structure (PDB code: 7AF1) in pink (backbone RMSD 0.48 Å). The 2Fo – Fc electron
density map (contoured at 0.6 in pymol) for the DNA is in grey mesh.
Using the crystallographically observed DNA as a tem- The complementary strand interacts with the protein via
plate we were able make a model for Artemis binding to a backbone contacts that span a 4-nucleotide sequence be-
longer section of double-stranded DNA by complementing tween 5- and 8-bases from the 3 -terminus with positively
unpaired bases on the single-stranded DNA overhang with charged sidechains in the MBL domain (Lys36, Lys40,
canonical base pairs, whilst maintaining acceptable geome- Arg43 and Lys74) (Figure 4C). The 3 -end of this strand
try of the sugar phosphate backbone (Figure 4A). The du- apparently terminates directly above a cluster of polar or
plex section of this model deviates slightly from the ideal B- positively charged residues in the -CASP domain (Lys207,
form geometry (57), in a manner that is reminiscent of some Lys288, Asn205) (Figure 4D). Whilst the experimental (as
transcription factor DNA complexes (58,59). We have also used in co-crystallization) DNA substrate and our model
modelled an extension of the metal ion contacting strand by both contain a 3 -hydroxyl group, the model implies that an
three nucleotides to form a 5 -overhang; the positioning of additional 3 -phosphate could be accommodated and may
the overhang nucleotides is more speculative, nevertheless it be expected to make favourable interactions with the ba-
was possible to avoid clashes with residues whist maintain- sic cluster of residues. Thus, our model suggests Artemis
ing a relaxed geometry (Figure 4E). preferentially binds DNA with a 5 -overhang binding at the
In the extended DNA complex model, Artemis contacts junction between double- and single-stranded regions; the
both strands of the DNA in several areas; notably a single expected product of this reaction would be a blunt ended
phosphate lies above the di-metal ion bearing active site and DNA with a 5 -phosphate. In the case of hairpin DNA sub-
ligates to both metal ions in the same manner as in struc- strates our model indicates Artemis may accommodate a
tures of related SNM1 enzymes (Baddock et al. 2021, ac- 4-nucleotide loop connecting the two paired strands, with
companying paper) (60). The two downstream nucleotides the cleavage product being DNA with a 3 -overhang cleaved
on this strand pass close to the protein surface, forming pos- from the last paired base of the duplex.
sible interactions with the backbone NH of Asp37 and the
sidechain of Lys36; subsequent nucleotides are not close to
Comparison of the Artemis DNA binding mode with that of
the protein (Figure 4C). The overhang portion of this strand
other nucleases
continues with a slightly altered trajectory, potentially con-
tacting Artemis in the vicinity of the cleft separating the In an accompanying paper [Baddock et al. 2021] we re-
MBL and -CASP domains, with the possibility of forming port a structure of SNM1B/Apollo in complex with two
favourable interactions with both positively charged (Arg deoxyadenosine monophosphate nucleotides and suggest
172) and aromatic residues (Phe173, Trp293) (Figure 4E). a model for SNM1B binding to DNA containing a 3 -
The surface between the MBL and -CASP domains of overhang (one of its preferred substrates). The overall mode
Artemis contains a belt of positively charged residues (Fig- of DNA binding is similar in the two models (Figure 4A
ure 4A), which likely facilitates productive DNA binding. and B), with the two DNA duplexes being roughly paral-9318 Nucleic Acids Research, 2021, Vol. 49, No. 16
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Figure 4. Proposed interactions of DNA with SNM1C/Artemis. Comparison of electrostatic surface potentials of DNA bound model for (A). Artemis
and (B). Apollo/SNM1B [Baddock et al. 2021 accompanying paper]. Blue colouring represents a more electropositive surface potential and red show a
more electronegative cluster. The active site contains two metal ions represented as a grey (zinc), orange (iron) and green (nickel) spheres. N- and C- termini
are in red. Electrostatic surface potentials were generated using PyMOL (electrostatic range: ±5). (C) A row of positively charged residues is on the MBL
domain surface of interact with the DNA phosphate backbone. (D) A DNA overhang is located at the active site. A cluster of polar residues (N205, K207
and K288) is located in the -CASP domain. The extended flexible loop, which is unique to Artemis compared to SNM1A and SNM1B, connecting G
and H is labelled. (E) The DNA overhang forms a hydrogen bond with Arg172 and interacts with a cluster of hydrophobic residues at the interface of
the MBL and -CASP domains.Nucleic Acids Research, 2021, Vol. 49, No. 16 9319
lel and forming contacts to similar regions on the MBL do- at the same concentration. We observed no exonuclease ac-
main. The most important differences lie in the nature of the tivity for the truncated Artemis construct, it is possible that
contacts formed to the active site and the paths of the vari- phosphorylation alters the balance between endonuclease
ous overhangs. In the SNM1B model, extensive contacts are and exonuclease activity, though the biological relevance of
made to the 5 -phosphate in a well-defined phosphate bind- this, if any, remains to be validated.
ing pocket. Both SNM1A and SNM1B are exclusively 5 - Both human SNM1A and SNM1B require a 5 -
phosphate exonucleases, with most of these phosphate bind- phosphate for activity (3,11,17). To investigate whether
ing residues being conserved (Supplementary Figure S2). there is a similar requirement for Artemis, we tested the
Artemis lacks these key phosphate binding residues and the activity of truncated Artemis against single-stranded and
5 -phosphate binding pocket of SNM1A and SNM1B. In- overhang DNA substrates with different 5 -end groups, in-
stead, in Artemis this pocket is partially filled by the Phe318 cluding a phosphate, hydroxyl and biotin groups (Figure
sidechain, which may inhibit the binding of the substrate 5 - 5A). The results obtained imply that, at least under the
phosphate. These differences rationalize major differences tested conditions, Artemis is agnostic to the different end
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in nuclease activities within the family, i.e. SNM1A and modifications, exhibiting comparable digestion of all sub-
SNM1B are exonucleases and Artemis is an endonuclease. strates.
Further differences between Artemis and Extensive evidence demonstrates that full-length Artemis
SNM1B/SNM1A are found in the loop connecting in complex with DNA-PKcs is a structure-selective endonu-
-strands G and H (using Artemis numbering), which in clease (19,20,29). These studies report Artemis can digest
Artemis is significantly longer and occupies a different po- substrates including overhangs, hairpins, stem-loops, and
sition contacting residues in the MBL domain (Figure 4D), splayed arms (pseudo-Y). To investigate the activity of trun-
compared to the loops in SNM1B and SNM1A that form cated Artemis catalytic domain (aa 1–361) we performed
part of the phosphate binding pocket and make potential assays using a variety of radio-labelled DNA substrates
contacts with the 3 -overhang. This loop displacement (Figure 5B). The results show that truncated Artemis has
in Artemis may contribute to its ability to accommodate substrate-selective endonuclease activity, with a preference
substrates with either 5 -overhangs or hairpins. for single-stranded DNA substrates, and those that con-
Another striking difference, at least in the available struc- tain single stranded character (e.g. 5 - and 3 -overhangs,
tures, is that the active site of Artemis is more open com- splayed arms, and a lagging flap structure), compared with
pared to those of SNM1A or SNM1B. This openness may double-stranded DNA structures (e.g. ds DNA and a repli-
reflect an ability to accommodate different substrate con- cation fork). This is in accord with previous reports, where
formations including hairpins, 3 - and 5 -overhangs, as well Artemis is reported to cleave around ss- to dsDNA junc-
as DNA flaps and gaps. Both human SNM1A and SNM1B tions in DNA substrates (62). The truncated Artemis cat-
appear to have a more sequestered active site that would alytic domain also exhibits hairpin opening activity, in ac-
only fit a single strand of DNA, which is consistent with pre- cordance with what has previously been reported (Supple-
vious findings on their preferred substrate selectivity (10). mentary Figure S9). On a duplex substrate (YM117 from
Ma et al.) (20) with a 20 nt hairpin region, Artemis cleaves
adjacent to the hairpin, consistent with previous data. It is
Biochemical characterization of truncated Artemis catalytic
clear that truncated form of Artemis exhibits nuclease ac-
domain (aa 1–361)
tivity closely comparable to the phosphorylated full-length
To investigate the activity of different versions of Artemis, Artemis protein (20), indicating that the structural stud-
we performed assays using radiolabelled DNA. We com- ies presented here reveal mechanistic insights of direct rel-
pared the catalytic domain purified using IMAC (which evance to the DNA-PKcs-associated form of Artemis that
contained Ni2+ in the active site) with catalytic domain pro- engages in end-processing reactions in vivo.
tein purified using ion exchange chromatography (avoid-
ing IMAC), which contained predominantly Zn2+ . We also
Structural and biochemical characterization of Artemis point
tested the activity of full-length phosphorylated Artemis.
mutations
The results show that both truncated enzymes have simi-
lar activity to the full-length enzyme (Supplementary Fig- Previous mutagenesis studies targeting metal ion coordinat-
ure S6). ing residues (D17N, H33A, H35A, D37N) of full-length
One notable difference between our full-length protein Artemis (aa 1–692) established the importance of motifs 1–
and that of Ma et al. (20), is that our protein is active in the 4 for activity (29). Each of these substitutions markedly re-
absence of DNA-PKcs. Intact mass spectrometric analysis duced or abolished Artemis’ ability to carry out its role in
of full-length protein shows that it has undergone up to five V(D)J recombination in vitro. We crystallized three forms of
phosphorylations (Supplementary Figure S7). Poinsignon truncated Artemis (aa 1–361) with substitutions in several
et al. have shown that Artemis is constitutively phospho- of these metal ion co-ordinating residues, i.e. D37A, H33A,
rylated in cultured mammalian cells and is further phos- and the Omenn Syndrome patient mutation H35D (33,56).
phorylated in response to induced DNA damage (61); it is The overall architecture of the three variants is almost iden-
interesting that the capacity to phosphorylate Artemis to tical to WT Artemis (Figure 6A). The D37A structure crys-
produce an active form is also conserved in insect cells. We tallizes with only one nickel ion (M1) (Figure 6B); Both the
observed exonuclease activity with full-length Artemis at H33A and H35D variants exhibited loss of the M1 ion (Fig-
10 nM enzyme concentration (Supplementary Figure S8), ure 6C and D). By contrast, all three variants retained the
though this was weak compared to its endonuclease activity Zn ion in the zinc finger-like motif of the -CASP domain.9320 Nucleic Acids Research, 2021, Vol. 49, No. 16
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Figure 5. Nuclease assay of truncated Artemis (aa 3–361) with various DNA substrates. (A) The nuclease activity of Artemis is indifferent to the 5´ group,
indicative of its endonuclease activity. Increasing concentrations of Artemis from 0 (NE; no enzyme) to 250 nM incubated with 10 nM ssDNA with either a
5 phosphate, 5 hydroxyl, or 5 biotin moiety (45 min, 37◦ C). (B) Artemis cleaves DNA substrates containing single-stranded regions. Increasing amounts
of Artemis were incubated with structurally diverse DNA substrates (10 nM) (45 min, 37◦ C). Products were analysed by 20% denaturing PAGE. The DNA
substrates utilized are represented at the top of the lanes and a red asterisk indicates the position of the 3 radiolabel.
Our D37A, H33A, and H35D variants lost the ability to ␣-helix E shifted upward and away from the zinc finger
digest DNA substrate in vitro (Figure 6E), in accord with like motif. These conformational changes suggest induced
results with full-length variants (29,33). fit during substrate binding. The combined results with the
Differential scanning fluorimetry (DSF) analyses showed variants thus reveal the importance of the HxHxDH motif
that both H33A and H35D variants are substantially desta- and highlight the importance of the di-metal catalytic core
bilized with Tm around -13◦ C compared to WT Artemis in the SNM1 family, not only in directly catalysing hydrol-
(Supplementary Figure S10). By contrast the D37A variant ysis, but also likely in conformational changes involved in
has similar thermal stability as the WT Artemis, suggest- catalysis (46).
ing that it is folded in the presence of a single metal ion in
the active site. Asp37 can adopt two conformations and the
Identification of small molecule Artemis inhibitors
coordinated zinc ion is present at about 30% occupancy in
both of the WT Artemis structures (PDB 6TT5 and 7AF1). Artemis, along with SNM1A and SNM1B, possess a con-
It is thus unsurprising that mutation of Asp37 to alanine served MBL-fold domain similar to that of the true bac-
results in the loss of Zn2. terial MBLs, suggesting that they may be inhibited by -
Histidines 33 and 35 are the first two histidine residues lactams. Studies on SNM1A/SNM1B, showed that ceftri-
in the HxHxDH motif (M1 binding) in the active site. In axone (Rocephin), a widely used -lactam (third generation
the absence of active site metal ions, the loop comprising cephalosporin) inhibits SNM1A/SNM1B (63). To investi-
residues 113–119 moves away from the active site (Figure gate if it inhibits Artemis we performed assays with ceftri-
6C and D) and a small rearrangement occurs in ␣8 (residues axone, cefotaxime, and 7-aminocephalosporanic acid (Fig-
348–358). In both the H33A and H35D variants, ␣8 moves ure 7A). The results show that neither cefotaxime nor 7-
slightly closer towards 14, compared to the WT and D37A aminocephalosporanic acid inhibit Artemis, whilst ceftriax-
variant. Larger differences occur in -strand E (residues one inhibits with a modest IC50 of 65 M (Figure 7B). We
268–270) and ␣-helix E (residues 261–267), both located solved a structure of ceftriaxone bound to Artemis (puri-
near the zinc finger motif in the -CASP domain (Figure fied by IMAC) at 1.9 Å resolution (Figure 7C) in the space
6A). In H33A and H35D variants, both -strand E and group P1 with one protein molecule in the asymmetric unit.Nucleic Acids Research, 2021, Vol. 49, No. 16 9321
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Figure 6. Structures of SNM1C/Artemis D37A, H33A and H35D variants and their activities. (A) Overlay of WT Artemis (PDB:7AF1, pink), and the
D37A (PDB:7AFS, yellow), H33A (PDB:7AFU, cyan) and H35D clinical (in Omen syndrome) (PDB:7AGI, blue) variants showing the overall folds are
conserved. The nickel ion is a green sphere and zinc ions are grey spheres. Movement of ␣E in the -CASP domain is indicated by a red arrow. (B) Left:
The active site of D37A variant has a single nickel ion (M1 in green); Right: active site residues of WT Artemis (pink), superimposed with that of the D37A
variant (yellow)––aside from loss of the second metal (M2) in the latter, there is little difference. (C) Active site of the H33A variant (cyan; left) and an
overlay (right) with WT Artemis (pink). The H33A variant is characterized by a lack of metal ions and a different conformation of the loop containing
His115. (D) The active site of the H35D variant (blue; left) and an overlay (right) with WT Artemis (pink). These variant lacks both metal ions, as for the
H33A variant. E. Comparing the activity of Artemis variants vs WT protein. Increasing amounts (from 0 to 250 nM) of WT and mutant Artemis proteins
(were incubated with 10 nM of 51 nucleotide ssDNA substrate (30 min, 37◦ C). Products were analysed by 20% denaturing PAGE.9322 Nucleic Acids Research, 2021, Vol. 49, No. 16
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Figure 7. Structural basis of SNM1C/Artemis inhibition by -lactam antibiotics. (A) Structures of selected -lactam antibiotics. (B) Effects of -lactams
on Artemis activity assessed via a real-time fluorescence-based nuclease assay. (C) Cartoon representation of the structure of truncated Artemis (aa1-361)
with ceftriaxone (white) bound at the active site (PDB: 7APV). (D) Active site residues with the electron density (Fo -Fc ) contoured map around the modelled
ceftriaxone. The map is contoured at the 1.0 level and was calculated before ceftriaxone was included in the refinement.
As before, in this structure Artemis possesses the canonical molecule. The rest of the molecule appears to be flexible.
bilobar MBL and -CASP fold with an active site contain- The binding mode of ceftriaxone to Artemis shown in Fig-
ing one nickel ion, likely reflecting the purification method. ure 7C is near identical to that observed for ceftriaxone with
Ceftriaxone binds to the protein surface in an extended SNM1A (PDB: 5NZW) structure (Supplementary Figure
manner making interactions with the active site, towards the S11).
-CASP domain (Figure 7C). There is no evidence for cleav- A notable difference between the ceftriaxone-bound
age of the -lactam, nor of loss of the C-3 cephalosporin Artemis structure and the non-complexed structure is the
side chain, reactions that can occur during ‘true’ MBL loss of a metal ion at M2 site, normally coordinated by
catalysed cephalosporin hydrolysis. Electron density at the Asp37, His38 and Asp130 (Figure 7D). Without a metal
active site clearly reveals the ceftriaxone side chain coor- ion at the M2 site, as in the ceftriaxone bound structure,
dinates the M1 site nickel ion replacing two waters (72 the Asp37 side chain is positioned away from the active site
and 106) in the uncomplexed structure (Figure 7D). De- (Figure 7D), as seen when nickel is bound in the M1 site
spite the conservation of key elements of the active site (Figure 1I).
of the MBL fold nucleases and the ‘true’ −lactam hy- To investigate the possibility of inhibiting Artemis
drolysing MBLs (64), ceftriaxone, does not interact with the through binding to the zinc finger motif in the -CASP do-
nickel ion via its -lactam carbonyl (as occurs for the true main, we used the fluorescence-based nuclease assay to test
MBLs), but via both carbonyl oxygens of the cyclic 1, 2 di- compounds known to react with thiol groups present in zinc
amide in its sidechain (Ni–O distances: 2 and 2.2 Å), i.e. fingers and which result in zinc ion displacement, i.e. ebse-
it is not positioned for productive -lactam hydrolysis. The len (65), auranofin (66) and disulfiram (67). Both ebselen
amino-thiazole group (N7) of ceftriaxone forms hydrogen and disulfiram inhibit Artemis with IC50 values around 8.5
bonds with the side chain of Asn205, while the S1 of the M and 10.8 M respectively, whilst auranofin inhibits less
7-aminocephalosporanic acid core of the compound inter- potently (IC50 46 M) (Supplementary Figure S12), indi-
acts with the hydroxyl of Tyr212 through an ethylene glycol cating additional possible inhibitory strategies.Nucleic Acids Research, 2021, Vol. 49, No. 16 9323
DISCUSSION We solved structures of three Artemis variants D37A,
H33A and an Omenn syndrome patient mutation, H35D.
The DCLRE1C/Artemis gene was first discovered in stud-
Using gel-based nuclease assays, we showed that these vari-
ies of children with a radiosensitive form of severe com-
ants are inactive. Overall, the three variant structures are
bined immunodeficiency disease (RS-SCID) (68). Subse-
similar to the WT structure, even though H33A and H35D
quent work has shown Artemis is a structure-specific en-
entirely lack any metal ions in their active site, although zinc
donuclease involved in V(D)J recombination (18,19,69) and
was present in the zinc finger. Mutation of Asp37 to alanine
the c-NHEJ DNA repair pathway (4,23,70) (2). Our struc-
results in the loss of the second metal in the catalytic site,
tures of wild-type and catalytic site mutants of Artemis pro-
likely explaining the loss of activity, although the first metal
tein show that, like SNM1A and SNM1B/Apollo and the
ion is still present. Note that some MBL fold hydrolases uses
RNA processing enzyme CPSF73, Artemis has a typical
two metal ions (e.g. B1 and B3 subfamilies of the true MBLs
␣/-/␣ sandwich fold in its MBL domain and has a -
and RNase J1 from Bacillus subtilis) (Supplementary Fig-
CASP domain, the latter a characteristic of MBL fold nu-
ure S13A and C) (64,74) whereas others, sometimes with
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cleases. Both our structures and those of Karim et al (37)
apparently similar active sites, only use one metal ion at the
reveal a unique and conserved structural feature of Artemis
M1 site (e.g. the B2 subfamily of the true MBLs and RNase
in its -CASP domain that is not reported in other human
J2 from Staphylococcus epidermis) (64,75) (Supplementary
MBL enzymes, i.e. a conserved (in Artemis orthologues)
Figure S13B and D). Thus, whilst our results support the
classical zinc-finger like motif.
importance of having both metals for the nuclease activity
Zinc-finger motifs are common in DNA binding proteins
by Artemis, subtle features can influence MBL fold enzyme
such as transcription factors (51,55) and are observed in re-
activity (64,71).
quired and accessory of NHEJ proteins (53). They are re-
Following re-analysis of the Karim et al. structure (PDB
ported to both provide stability and enhance substrate se-
code 6WO0), we generated a model of a DNA overhang in
lectivity rather than being directly involved in catalysis; we
complex with Artemis that informs on the substrate bind-
propose that this is likely the case for Artemis. Further-
ing mode. Our model shows that Artemis interacts with
more, point mutations in His 228 and His 254 (H228N and
its DNA substrate at the interface between the MBL and
H254L) have been reported in patients with a SCID pheno-
the -CASP domains. This interaction is mediated through
type (56). The role of the zinc-finger motif in Artemis is un-
a combination of polar or positive residues and aromatic
known and the subject of ongoing investigations. We have
residues of Artemis and the DNA substrate (Figure 6). A
mutated the zinc finger residues (His 228, His 254 Cys 256
related interaction was also observed in a cryo-EM struc-
and Cys 272), but none of the mutated constructs produced
ture of the pre-mRNA processing enzyme CPSF-73 with
purified protein. Although inconclusive, this may indicate
substrate bound, where residues R34, R174, K24 and F176
that the Zinc finger is important for the structural integrity
(CPSF-73 numbering) form direct contacts with the sub-
or stability of the protein.
strate (76). The Artemis DNA bound structure (PDB code
The presence of one or two metal ions coordinated by
7ABS) contains a 2-base pair region of duplex DNA that
the HxHxDH motif at the Artemis active site is a hallmark
makes close contact with the metal centre in the Artemis
of the SNM1 family (11,71); the available evidence implies
active site, which could potentially be catalytically relevant
that metal ion binding at one site (M1 site in standard MBL
(Figure 3). This phosphate ion coordinates the di-metal cen-
nomenclature) is stronger than at the other (M2 site). By
tre in a similar manner to the AMP molecule bound to
analogy with studies on the true MBLs, these metal ions
the active site of SNM1B/Apollo [Baddock et al. 2021, ac-
are proposed to activate a water / hydroxide ion that act as
companying paper], and the phosphate backbone of the
the nucleophile in the phosphodiester cleavage (72). This hy-
pre-mRNA substrate bound in CPSF-73 (PDB code 6V4X)
droxide ion is also present in the crystal structure of CPSF-
(76). By analogy with proposals for true MBLs (71,77), the
73 (Figure 1G), along with an adjacent sulphate ion (47),
mechanism of SNM1A/B and Artemis catalysed phosphate
which is proposed to mimic the phosphate group of the pre-
hydrolysis thus likely involves metal ion mediated reaction
mRNA at the cleavage site. Our structure (PDB: 7AF1) sug-
of a hydroxide ion with the phosphate and activation of
gests that the native metal ion(s) residing in the M1 site
the phosphate by metal ion chelation. However, the precise
of Artemis is zinc, although a nickel ion can also occupy
identity of the in vivo relevant SNM1 active site metal ions
the M1 site depending on the how the protein was purified
and whether there are one or two metal ions is uncertain.
(PDB: 6TT5). Neither the presence of copurifying Ni ion in
The true MBLs can be active with two metal ions (B1 and
the active site, nor the truncation of the C-terminal tail, ap-
B2 subfamily MBLs) or one metal ion (B2 MBLs); these
pear to substantially inhibit the activity of Artemis. Thus,
metal ions are likely zinc ions, but even this has not been
using radio-labelled gel-based nuclease assays, we showed
unequivocally demonstrated within a human disease con-
that the truncated Artemis catalytic domain (aa 1–361) with
text and it is possible that metal ions other than zinc are
either copurifying Zn or Ni ions (as observed crystallo-
relevant (77,78). The available evidence is that the SNM1
graphically in the same preparations) have similar activity
MBL nucleases employ two (zinc) metal ions, with one (at
with the full-length Artemis construct (aa 1–693). There-
the M2 site) being less tightly bound than the other (M1
fore, it seems likely that nickel ions are able to replace zinc
site) – though considerable uncertainty remains as to the in
ions in solution; catalysis of MBL fold enzymes, including
vivo relevant metal ions. Binding of a metal ion at the M2
hydrolytic reactions, with metal ions other than zinc is well-
site appears to be promoted by the DNA substrate and it
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